Polymer conjugates of opioid antagonists

ABSTRACT

The invention provides polymer conjugates of opioid antagonists comprising a polymer, such as poly(ethylene glycol), covalently attached to an opioid antagonist. The linkage between the polymer and the opioid antagonist is preferably hydrolytically stable. The invention also includes a method of treating one or more side effects associated with the use of opioid analgesics, such as constipation, nausea, or pruritus, by administering a polymer conjugate of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Provisional Application Ser. No.60/330,400, filed Oct. 18, 2001, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to water-soluble polymer conjugates ofbiologically active molecules, and in particular, to water-solublepolymer conjugates of opioid antagonists, such as naloxone, and relatedpharmaceutical compositions and uses thereof.

BACKGROUND OF THE INVENTION

Natural and synthetic alkaloids of opium (i.e., opioids) are useful asanalgesics for the treatment of severe pain. Opioids target three typesof endogenous opioid receptors: μ-, δ-, and κ-receptors. Many opioids,such as morphine, are μ-receptor agonists that are highly efficaciousanalgesic compounds due to their activation of opioid receptors in thebrain and central nervous system (CNS). Opioid receptors are, however,not only limited to the CNS, but may be found in other tissuesthroughout the body. These receptors located outside the CNS arereferred to as peripheral receptors. A number of side effects associatedwith opioid use are caused by activation of these peripheral receptors.For example, administration of opioid agonists often results inintestinal dysfunction due to action of the opioid agonist upon thelarge number of receptors in the intestinal wall. Specifically, opioidsare generally known to cause nausea and vomiting as well as inhibitionof normal propulsive gastrointestinal function in animals, resulting inside effects such as constipation.

Opioid-induced side effects are a serious problem for patients beingadministered opioid analgesics for both short term and long term painmanagement. For instance, more than 250,000 terminal cancer patientseach year take opioids, such as morphine, for pain relief, and abouthalf of those patients experience severe constipation. In manysituations the discomfort can be so great that the patients choose toforego the pain relief in order to avoid the constipation. In an effortto address this problem, certain opioid antagonist compounds that do notreadily cross the blood-brain barrier have been tested for use incurbing opioid-induced side effects. For instance, the peripheralμ-opioid antagonist compound, methylnaltrexone, and related compoundshave been suggested for use in assuaging opioid-induced side effects.See for example, U.S. Pat. Nos. 5,972,954, 5,102,887, 4,861,781, and4,719,215, which describe the use of methylnaltrexone and relatedcompounds in controlling opioid-induced pruritus, nausea, and/orvomiting. Methylnaltrexone, however, is an experimental drug and is notcommercially available. Unfortunately, most of the currently availableopioid antagonists, such as the tertiary opioid antagonist, naloxone,are small molecules that not only possess antagonist activity atperipheral receptors associated with the intestine, but also possessantagonist activity at CNS receptors since they cross the blood-brainbarrier. Consequently, many opioid antagonists interfere with the painrelief brought about by administration of opioid-based analgesics. Thus,at present, patients receiving opioid pain medications face thedifficult choice of suffering burdensome adverse effects such asconstipation or ineffective analgesia.

Thus, there is a need in the art for alternative compounds, or forapproaches for modifying or improving upon existing compounds, that canreduce or eliminate opioid-induced side effects such as constipation,even when administered in high doses, without interfering with thepain-suppressing effects of the opioid.

SUMMARY OF THE INVENTION

The present invention is based upon the development of water-soluble,polymer-modified opioid antagonist compounds designed for the treatmentof opioid-induced side effects such as constipation, while not reversingor impacting analgesia.

In one aspect, the present invention provides a polymer conjugatecomprising a water-soluble and non-peptidic polymer covalently attachedto an opioid antagonist.

Suitable polymers for covalent attachment to an opioid antagonistinclude poly(alkylene glycols), poly(oxyethylated polyol), poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), poly(acrylic acid), carboxymethyl cellulose,hyaluronic acid, hydroxypropylmethyl cellulose, and copolymers,terpolymers, and mixtures thereof. In one embodiment of the invention,the polymer is a polyethylene glycol. In an alternative embodiment, thepolymer is polyacrylic acid.

The polymer portion of a conjugate of the invention may be linear, suchas methoxy PEG, branched, or forked. In particular embodiments of theinvention wherein the polymer is linear, the conjugate may incorporate aheterobifunctional or a homobifunctional polymer. A conjugate of aheterobifunctional polymer is one wherein one terminus of the polymerattached to the opioid antagonist and the other terminus isfunctionalized with a different moiety. A conjugate of ahomobifunctional polymer possesses a structure wherein each end of alinear polymer is covalently attached to an opioid antagonist, typicallyby an identical linkage.

Exemplary opioid antagonists include buprenorphine, cyclazocine,cyclorphan, naloxone, N-methylnaloxone, naltrexone, N-methylnaltrexone,nalmephene, 6-amino-6-desoxo-naloxone, levallorphan, nalbuphine,naltrendol, naltrindole, nalorphine, nor-binaltorphimine, oxilorphan,pentazocine, piperidine-N-alkylcarboxylate opioid antagonists, andopioid antagonist polypeptides. One particularly preferred opioidantagonist is naloxone or a derivative thereof, such as6-amino-6-desoxo-naloxone.

In yet another embodiment, the polymer conjugate is covalently attachedto the opioid antagonist by a hydrolytically stable linkage.Hydrolytically stable linkages include amide, amine, carbamate, ether,thioether, and urea-based linkages.

In one embodiment of the invention, the molecular weight of the polymeris less than about 5,000 daltons (Da).

In yet another embodiment, the molecular weight of the polymer is lessthan about 2,000 Da.

In yet an even more preferred embodiment, the molecular weight of thepolymer is less than about 1,000 Da.

In yet another embodiment, the molecular weight of the polymer is lessthan about 800 Da.

In another aspect, the invention encompasses a pharmaceuticalcomposition containing a polymer conjugate as described above incombination with a pharmaceutically acceptable carrier.

According to yet another aspect, the invention provides a method oftreating at least one side effect of opioid administration, particularlyside effects associated with the gastrointestinal system (e.g., nauseaand constipation) by administering a conjugate of a water-soluble andnon-peptidic polymer covalently attached to an opioid antagonist.

In one embodiment of the method, the conjugate is preferablyadministered conjointly with an opioid agonist, meaning the conjugate isadministered at the same time as the opioid agonist or within a shortperiod of time before or after administration of the opioid agonist. Inyet a further embodiment of the method, the conjugate is administeredorally.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

I. DEFINITIONS

The following terms as used herein have the meanings indicated.

As used in the specification, and in the appended claims, the singularforms “a”, “an”, “the”, include plural referents unless the contextclearly dictates otherwise.

The terms “functional group”, “active moiety”, “reactive site”,“chemically reactive group” and “chemically reactive moiety” are used inthe art and herein to refer to distinct, definable portions or units ofa molecule. The terms are somewhat synonymous in the chemical arts andare used herein to indicate the portions of molecules that perform somefunction or activity and are reactive with other molecules. The term“active,” when used in conjunction with a functional group, is intendedto include those functional groups that react readily with electrophilicor nucleophilic groups on other molecules, in contrast to those groupsthat require strong catalysts or highly impractical reaction conditionsin order to react (i.e., “non-reactive” or “inert” groups). For example,as would be understood in the art, the term “active ester” would includethose esters that react readily with nucleophilic groups such as amines.Exemplary active esters include N-hydroxysuccinimidyl esters or1-benzotriazolyl esters. Typically, an active ester will react with anamine in aqueous medium in a matter of minutes, whereas certain esters,such as methyl or ethyl esters, require a strong catalyst in order toreact with a nucleophilic group. As used herein, the term “functionalgroup” includes protected functional groups.

The term “protected functional group” or “protecting group” or“protective group” refers to the presence of a moiety (i.e., theprotecting group) that prevents or blocks reaction of a particularchemically reactive functional group in a molecule under certainreaction conditions. The protecting group will vary depending upon thetype of chemically reactive group being protected as well as thereaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule, if any. Protecting groupsknown in the art can be found in Greene, T. W., et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley & Sons, New York, N.Y.(1999).

The term “linkage” or “linker” (L) is used herein to refer to an atom ora collection of atoms used to link, preferably by one or more covalentbonds, interconnecting moieties such as two polymer segments or aterminus of a polymer and a reactive functional group present on abioactive agent, such as an opioid antagonist. A linker of the inventionmay be hydrolytically stable or may include a physiologicallyhydrolyzable or enzymatically degradable linkage.

A “physiologically hydrolyzable” or “hydrolytically degradable” bond isa weak bond that reacts with water (i.e., is hydrolyzed) underphysiological conditions. Preferred are bonds that have a hydrolysishalf life at pH 8, 25° C. of less than about 30 minutes. The tendency ofa bond to hydrolyze in water will depend not only on the general type oflinkage connecting two central atoms but also on the substituentsattached to these central atoms. Appropriate hydrolytically unstable ordegradable linkages include but are not limited to carboxylate ester,phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether,imines, orthoesters, peptides and oligonucleotides.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

An “enzymatically unstable” or degradable linkage is a linkage that canbe degraded by one or more enzymes.

The term “polymer backbone” refers to the covalently bonded chain ofrepeating monomer units that form the polymer. The terms polymer andpolymer backbone are used herein interchangeably. For example, thepolymer backbone of PEG is —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂ where ntypically ranges from about 2 to about 4000. As would be understood, thepolymer backbone may be covalently attached to terminal functionalgroups or pendant functionalized side chains spaced along the polymerbackbone.

The term “reactive polymer” refers to a polymer bearing at least onereactive functional group.

Unless otherwise noted, molecular weight is expressed herein as numberaverage molecular weight (M_(n)), which is defined as

$\frac{\sum{NiMi}}{\sum{Ni}},$

wherein Ni is the number of polymer molecules (or the number of moles ofthose molecules) having molecular weight Mi.

The term “alkyl”, “alkenyl”, and “alkynyl” refers to hydrocarbon chainstypically ranging from about 1 to about 12 carbon atoms in length,preferably 1 to about 6 atoms, and includes straight and branchedchains.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablycomprising 3 to about 12 carbon atoms, more preferably 3 to about 8.

The term “substituted alkyl”, “substituted alkenyl”, “substitutedalkynyl” or “substituted cycloalkyl” refers to an alkyl, alkenyl,alkynyl or cycloalkyl group substituted with one or more non-interferingsubstituents, such as, but not limited to, C3-C8 cycloalkyl, e.g.,cyclopropyl, cyclobutyl, and the like; acetylene; cyano; alkoxy, e.g.,methoxy, ethoxy, and the like; lower alkanoyloxy, e.g., acetoxy;hydroxy; carboxyl; amino; lower alkylamino, e.g., methylamino; ketone;halo, e.g. chloro or bromo; phenyl; substituted phenyl, and the like.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C1-C6 alkyl (e.g., methoxy or ethoxy).

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Multiple aryl rings may be fused, as in naphthyl or unfused, asin biphenyl. Aryl rings may also be fused or unfused with one or morecyclic hydrocarbon, heteroaryl, or heterocyclic rings.

“Substituted aryl” is aryl having one or more non-interfering groups assubstituents. For substitutions on a phenyl ring, the substituents maybe in any orientation (i.e., ortho, meta or para).

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof, which heteroaryl groupis optionally substituted at carbon or nitrogen atom(s) with C1-6 alkyl,—CF₃, phenyl, benzyl, or thienyl, or a carbon atom in the heteroarylgroup together with an oxygen atom form a carbonyl group, or whichheteroaryl group is optionally fused with a phenyl ring. Heteroarylrings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings. Heteroaryl includes, but is notlimited to, 5-membered heteroaryls having one hetero atom (e.g.,thiophenes, pyrroles, furans); 5-membered heteroaryls having twoheteroatoms in 1, 2 or 1,3 positions (e.g., oxazoles, pyrazoles,imidazoles, thiazoles, purines); 5-membered heteroaryls having threeheteroatoms (e.g., triazoles, thiadiazoles); 5-membered heteroarylshaving 3 heteroatoms; 6-membered heteroaryls with one heteroatom (e.g.,pyridine, quinoline, isoquinoline, phenanthrine,5,6-cycloheptenopyridine); 6-membered heteroaryls with two heteroatoms(e.g., pyridazines, cinnolines, phthalazines, pyrazines, pyrimidines,quinazolines); 6-membered heteroaryls with three heteroatoms (e.g.,1,3,5-triazine); and 6-membered heteroaryls with four heteroatoms.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand at least one ring atom which is not carbon. Preferred heteroatomsinclude sulfur, oxygen, and nitrogen. Multiple rings may be fused, as inquinoline or benzofuran.

“Substituted heterocycle” is heterocycle having one or more side chainsformed from non-interfering substituents.

“Non-interfering substituents are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

Suitable non-interfering substituents or radicals include, but are notlimited to, halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, C3-C10 cycloalkyl, C3-C10cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl,C2-C12 alkoxyalkyl, C7-C12 alkoxyaryl, C7-C12 aryloxyalkyl, C6-C12oxyaryl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl, —(CH₂)_(m)—O—(C1-C10alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substitutedalkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclicradical, nitroalkyl, —NO₂, —CN, —NRC(O)—(C1-C10 alkyl), —C(O)—(C1-C10alkyl), C2-C10 thioalkyl, —C(O)O—(C1-C10 alkyl), —OH, —SO₂, ═S, —COOH,—NR, carbonyl, —C(O)—(C1-C10 alkyl)-CF₃, —C(O)—CF₃, —C(O)NR₂, —(C1-C10alkyl)-S—(C6-C12 aryl), —C(O)—(C6-C12 aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C1-C10 alkyl) wherein each m is from 1 to 8,—C(O)NR, —C(S)NR, —SO₂NR, —NRC(O)NR, —NRC(S)NR, salts thereof, and thelike. Each R as used herein is H, alkyl or substituted alkyl, aryl orsubstituted aryl, aralkyl, or alkaryl.

“Heteroatom” means any non-carbon atom in a hydrocarbon analog compound.Examples include oxygen, sulfur, nitrogen, phosphorus, arsenic, silicon,selenium, tellurium, tin, and boron.

The term “drug”, “biologically active molecule”, “biologically activemoiety” or “biologically active agent”, when used herein means anysubstance which can affect any physical or biochemical properties of abiological organism, including but not limited to viruses, bacteria,fungi, plants, animals, and humans. In particular, as used herein,biologically active molecules include any substance intended fordiagnosis, cure mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals. Examples of biologically activemolecules include, but are not limited to, peptides, proteins, enzymes,small molecule drugs, dyes, lipids, nucleosides, oligonucleotides,polynucleotides, nucleic acids, cells, viruses, liposomes,microparticles and micelles. Classes of biologically active agents thatare suitable for use with the invention include, but are not limited to,antibiotics, fungicides, anti-viral agents, anti-inflammatory agents,anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones,growth factors, steroidal agents, and the like.

“Polyolefinic alcohol” refers to a polymer comprising a polyolefinbackbone, such as polyethylene, having multiple pendant hydroxyl groupsattached to the polymer backbone. An exemplary polyolefinic alcohol ispolyvinyl alcohol.

As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer backbonemay include a minor number of peptide linkages spaced along the lengthof the backbone, such as, for example, no more than about 1 peptidelinkage per about 50 monomer units.

“Polypeptide” refers to any molecule comprising a series of amino acidresidues, typically at least about 10-20 residues, linked through amidelinkages (also referred to as peptide linkages) along the alpha carbonbackbone. While in some cases the terms may be used synonymously herein,a polypeptide is a peptide typically having a molecular weight up toabout 10,000 Da, while peptides having a molecular weight above that arecommonly referred to as proteins. Modifications of the peptide sidechains may be present, along with glycosylations, hydroxylations, andthe like. Additionally, other non-peptidic molecules, including lipidsand small drug molecules, may be attached to the polypeptide.

By “residue” is meant the portion of a molecule remaining after reactionwith one or more molecules. For example, an opioid antagonist residue inthe polymer conjugate of the invention is the portion of an opioidantagonist remaining following covalent linkage to a polymer backbone.

“Oligomer” refers to short monomer chains comprising 2 to about 10monomer units, preferably 2 to about 5 monomer units.

The term “conjugate” is intended to refer to the entity formed as aresult of covalent attachment of a molecule, e.g., a biologically activemolecule such as an opioid antagonist, to a reactive polymer molecule,preferably poly(ethylene glycol).

“Bifunctional” in the context of a polymer of the invention refers to apolymer possessing two reactive functional groups which may be the sameor different.

“Multifunctional” in the context of a polymer of the invention means apolymer having 3 or more functional groups attached thereto, where thefunctional groups may be the same or different. Multifunctional polymersof the invention will typically comprise from about 3-100 functionalgroups, or from 3-50 functional groups, or from 3-25 functional groups,or from 3-15 functional groups, or from 3 to 10 functional groups, orwill contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups attached to thepolymer backbone.

II. POLYMER CONJUGATES OF OPIOID ANTAGONISTS

As described generally above, the polymer conjugates of the inventioncomprise a water-soluble and non-peptidic polymer covalently attached toan opioid antagonist. The polymer conjugates of the invention are usefulfor the treatment of one or more side effects of opioid analgesicadministration, such as nausea, pruritus or constipation. The conjugatesof the invention typically comprise a polymer having a molecular weightselected such that the conjugate either i) does not pass to anyappreciable extent through the intestinal wall and into the bloodstream,so as to increase the localized concentration of polymer conjugate inthe intestine and promote binding to opioid receptors in the intestinalwall, and/or ii) does not pass through the blood-brain barrier and intothe CNS. According to one feature of the invention, upon administration,the polymer conjugate is retained within the gastrointestinal system andacts directly in the gut, or at least outside of the CNS, to reduce thelikelihood of the opioid antagonist interfering with the analgesiceffects of the opioid compound. In this manner, the polymer conjugatesof the invention are capable of treating the common side effects ofopioid use by selectively reacting with peripheral receptors withoutadversely impacting the analgesic effect of the opioid.

So, in essence, covalent attachment of the polymer to the opioidantagonist can increase the resistance of the conjugate to bothintestinal barrier transport (e.g., into the circulation) andblood-brain barrier transport as compared to the unmodified opioidantagonist, thereby (i) preventing the opioid antagonist frominterfering with the pain relief provide by the opioid and (ii)improving the effectiveness of the unmodified opioid antagonist.

For the most effective treatment of opioid-induced constipation stemmingfrom interaction of the opioid with opioid receptors within theintestinal wall, it is preferable to select a polymer molecular weightthat prevents or at least significantly reduces penetration of thepolymer conjugate through the intestinal wall and into the bloodstream.Preferably, the molecular weight of the polymer is selected so as not toimpede penetration of the polymer conjugate into the mucosal membrane ofthe intestinal barrier. As would be understood, the mucosal membrane isthe primary intestinal barrier to potentially harmful antigens andbacteria and comprises epithelial cells that secrete, and are coatedwith, a layer of mucus about 2 mm thick, which adheres tightly to thecell membranes. The mucus lubricates the epithelial cell surfaces andprevents mechanical damage by the stomach contents. Although not boundby any particular theory, penetration into the mucosal membrane isbelieved to promote interaction between the polymer conjugate of theinvention and the peripheral opioid receptors in the intestinal wall.Thus, the conjugates of the invention are preferably designed to achievea balance of factors, such as (i) maintaining the antagonist activity ofthe opioid antagonist, (ii) penetrating the mucosal barrier of theintestine while not crossing to a significant extent from the intestineinto the bloodstream, and (iii) if present in the general circulation,exhibiting the inability to cross the blood-brain barrier to anysignificant degree.

Typically, the number average molecular weight of the polymer portion ofa polymer conjugate of the invention is less than about 5,000 daltons(Da), and more preferably is less than about 2,000 Da. In an even morepreferred embodiment of the invention, the polymer possesses a molecularweight of about 1,000 Da or less, or of about 800 Da or less. In turningnow to ranges of molecular weights for the polymer portion of theconjugate, the molecular weight range is generally from about 100 Da toabout 2,000 Da, preferably about 100 Da to about 1,000 Da, morepreferably about 100 Da to about 800 Da, or from about 100 Da to about500 Da. Polymer backbones having a number average molecular weight ofabout 100 Da, about 200 Da, about 300 Da, about 400 Da, about 500 Da,about 550 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da andabout 1,000 Da are particularly preferred. The polymers of the inventionare hydrophilic in nature, thereby imparting hydrophilicity to theresulting conjugates and making them unable to cross the blood-brainbarrier to a significant extent.

To reduce the possibility of deactivation of the antagonist activity ofthe opioid antagonist compound and to keep the total molecular weight ofthe polymer backbone portion of the conjugate within the preferredrange, it is sometimes preferable to only attach a single polymerbackbone to the opioid antagonist molecule, or, if employing a branchedpolymer, to utilize a polymer at the lower end of the preferredmolecular weight ranges described above. Alternatively, a linear orforked polymer having two opioid antagonist molecules attached may beused to achieve the desired balance of activity and penetrationcharacteristics.

The linkage between the polymer backbone and the opioid antagonist ispreferably hydrolytically stable so that the opioid antagonist is notreleased from the polymer following administration to a patient. Releaseof the opioid antagonist in vivo could lead to a loss in analgesiceffect of the opioid compound due to passage of the released opioidantagonist into the CNS. Representative linkages for connecting theopioid antagonist and the polymer include ether, amide, urethane (alsoknown as carbamate), amine, thioether (also known as sulfide), and urea(also known as carbamide) linkages. The particular linkage and linkagechemistry employed will depend upon the subject opioid antagonist,functional groups within the molecule available either for attachment toa polymer or conversion to a suitable attachment site, the presence ofadditional functional groups within the molecule, and the like, and canbe readily determined by one skilled in the art based upon the guidancepresented herein.

The polymer conjugates of the invention maintain at least a measurabledegree of specific opioid antagonist activity. That is to say, a polymerconjugate in accordance with the invention will possesses anywhere fromabout 1% to about 100% or more of the specific activity of theunmodified parent opioid antagonist compound. Such activity may bedetermined using a suitable in-vivo or in-vitro model, depending uponthe known activity of the particular opioid antagonist parent compound.For example, a hot plate or tail flick analgesia assay can be used toassess the level of antagonist activity of the polymer conjugates of theinvention (See, for example, Tulunay, et al., J Pharmacol Exp Ther 1974;190:395-400; Takahashi, et al., Gen Pharmacol 1987; 18(2):201-3;Fishman, et al., Pharmacology 1975; 13(6):513-9). In general, a polymerconjugate of the invention will possess a specific activity of at leastabout 2%, 5%, 10%, 15%, 25%, 30%, 40%, 50%, 60%, 80%, 90% or morerelative to that of the unmodified parent opioid antagonist, Whenmeasured in a suitable model, such as those well known in the art.Preferably, a conjugate of the invention will maintain at least 50% ormore of the opioid antagonist activity of the unmodified parentcompound.

In addition to maintaining at least a portion of the opioid antagonistactivity of the parent opioid antagonist compound, the polymerconjugates of the invention also exhibit high levels of activity withrespect to peripheral opioid receptors in gastrointestinal tissue, whileexhibiting substantially no activity with respect to opioid receptors inthe CNS. The term “substantially no CNS activity”, as used herein, meansthe polymer conjugates of the invention cause less than about a 25%reduction in the analgesic effect of the opioid agonist, which can bemeasured, for example, using a tail flick or hot plate analgesia assayas described above. In preferred embodiments, the polymer conjugates ofthe invention cause less than about 20% reduction in the analgesiceffect of the opioid agonist, more preferably less than about 15%reduction, or even less than about 10% or less than about 5% reduction.A reduction of analgesic effect of about 0% (i.e., no reduction inanalgesia) is most preferred.

A polymer conjugate of the invention will typically comprise awater-soluble and non-peptidic polymer, such as poly(ethylene glycol),covalently attached to an opioid antagonist and having a generalizedstructure as shown below.

POLY-X-A_(o)  Formula I

wherein:

POLY is a water-soluble and non-peptidic polymer;

X is a linkage, preferably a hydrolytically stable linkage covalentlyattaching the polymer to the opioid antagonist; and

A_(o) is the opioid antagonist.

In one preferred embodiment, the conjugate of Formula I has thestructure:

wherein:

Y is C1-C6 alkyl, substituted C1-C6 alkyl, C3-C6 cycloalkyl, substitutedC1-C6 cycloalkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6alkynyl, substituted C2-C6 alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocycle, and substituted heterocycle;

Z is H or OH;

the dashed line indicates an optional double bond; and

X and POLY are as defined above.

In another embodiment, the conjugate of Formula I has the structure:

wherein:

R₁ and R₂ are each independently hydrogen or OH, or together form ═CH₂or ═O; and

X, Y, Z, the dashed line, and POLY are as defined above.

In either Formula Ia or Formula Ib, preferred Y groups include C1-C6alkyl, substituted C1-C6 alkyl (e.g., C1-C6 alkyl substituted with C1-C6cycloalkyl), C2-C6 alkenyl (e.g., allyl), substituted C2-C6 alkenyl(e.g., chloroallyl), C2-C6 alkynyl (e.g., propargyl), substituted C2-C6alkynyl, C3-C6 cycloalkyl, and substituted C3-C6 cycloalkyl.

As would be understood, the particular Y, Z, R₁, and R₂ groups employedwill depend on the specific opioid antagonist used to form the polymerconjugate of the invention. Preferred opioid antagonists includenaloxone or derivatives thereof (i.e., Y=allyl, Z=OH, R₁ and R₂ togetherform ═O, no optional double bond), nalbuphine or derivatives thereof(i.e., Y=(cyclobutyl)methyl, Z=OH, R₁=H, R₂=0H, no optional doublebond), nalmephene or derivatives thereof (i.e., Y=(cyclopropyl)methyl,Z=OH, R₁ and R₂ together form ═CH₂, no optional double bond), naltrexoneor derivatives thereof (i.e., Y=(cyclopropyl)methyl, Z=OH, R₁ and R₂together form ═O, no optional double bond), and nalorphine orderivatives thereof (Y=allyl, Z=H, R₁=H, R₂=OH, optional double bondpresent).

The polymer conjugates of the invention may be administered per se or inthe form of a pharmaceutically acceptable salt, and any reference to thepolymer conjugates of the invention herein is intended to includepharmaceutically acceptable salts. If used, a salt of the polymerconjugate should be both pharmacologically and pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare the free active compound or pharmaceuticallyacceptable salts thereof and are not excluded from the scope of thisinvention. Such pharmacologically and pharmaceutically acceptable saltscan be prepared by reaction of the polymer conjugate with an organic orinorganic acid, using standard methods detailed in the literature.Examples of useful salts include, but are not limited to, those preparedfrom the following acids: hydrochloric, hydrobromic, sulfuric, nitric,phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric,citric, methanesulphonic, formic, malonic, succinic,naphthalene-2-sulphonic and benzenesulphonic, and the like. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium, or calcium salts of acarboxylic acid group.

A. Polymer Backbone

In general, the water soluble and non-peptidic polymer portion of theconjugate should be non-toxic and biocompatible, meaning that thepolymer is capable of coexistence with living tissues or organismswithout causing harm. When referring to a polymer conjugate, it is to beunderstood that the polymer can be any of a number of water soluble andnon-peptidic polymers, such as those described herein as suitable foruse in the present invention. Preferably, poly(ethylene glycol) (PEG) isthe polymer backbone. The term PEG includes poly(ethylene glycol) in anyof a number of geometries or forms, including linear forms (e.g., alkoxyPEG or bifunctional PEG), branched or multi-arm forms (e.g., forked PEGor PEG attached to a polyol core), pendant PEG, or less preferably, PEGwith degradable linkages therein, to be more fully described below.

In its simplest form, PEG has the formula

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—  Formula II

wherein n is from about 2 to about 45, typically from about 2 to about20.

As described above, end-capped polymers, meaning polymers having atleast one terminus capped with a relatively inert group (e.g., an alkoxygroup), can be used as a polymer of the invention. For example,methoxy-PEG-OH, or mPEG in brief, is a form of PEG wherein one terminusof the polymer is a methoxy group, while the other terminus is ahydroxyl group that is subject ready chemical modification. Thestructure of mPEG is given below,

CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH  Formula III

-   -   wherein n is as described above.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the PEG polymer. Generally speaking, amulti-armed or branched polymer possesses two or more polymer “arms”extending from a central branch point (e.g., C in the structure below)that is covalently attached, either directly or indirectly viaintervening connecting atoms, to one active moiety such as an opioidantagonist. For example, an exemplary branched PEG polymer can have thestructure:

wherein:

-   -   poly_(a) and poly_(b) are PEG backbones, such as methoxy        poly(ethylene glycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine.

The PEG polymer may alternatively comprise a forked PEG. Generallyspeaking, a polymer having a forked structure is characterized as havinga polymer chain attached to two or more active agents via covalentlinkages extending from a hydrolytically stable branch point in thepolymer. An example of a forked PEG is represented by PEG-YCHZ₂, where Yis a linking group and Z is an activated terminal group, such as analdehyde group, for covalent attachment to an opioid antagonist, linkedto CH by a chain of atoms of defined length. International ApplicationNo. PCT/US99/05333, the contents of which are incorporated by referenceherein, discloses various forked PEG structures capable of use in thepresent invention. The chain of atoms linking the Z functional groups tothe branching carbon atom serve as a tethering group and may comprise,for example, an alkyl chain, ether linkage, ester linkage, amidelinkage, or combinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG backbone rather than at the end of the PEG chain. The pendantreactive groups can be attached to the PEG backbone directly or througha linking moiety, such as an alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the polymerbackbone, including any of the above described polymers, although thisembodiment is somewhat less preferred for the conjugates of the presentinvention. For example, PEG can be prepared with ester linkages in thepolymer backbone that are subject to hydrolysis. As shown below, thishydrolysis results in cleavage of the polymer into fragments of lowermolecular weight:

-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al., Polymer Preprints, 38(1):582-3 (1997), which isincorporated herein by reference.); phosphate ester linkages formed, forexample, by reacting an alcohol with a phosphate group; hydrazonelinkages which are typically formed by reaction of a hydrazide and analdehyde; acetal linkages that are typically formed by reaction betweenan aldehyde and an alcohol; ortho ester linkages that are, for example,formed by reaction between a formate and an alcohol; peptide linkagesformed by an amine group, e.g., at an end of a polymer such as PEG, anda carboxyl group of a peptide; and oligonucleotide linkages formed by,for example, a phosphoramidite group, e.g., at the end of a polymer, anda 5′ hydroxyl group of an oligonucleotide.

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms of PEG.

As noted previously above, any of a variety of monofunctional,bifunctional or multifunctional polymers that are non-peptidic andwater-soluble can also be used to form a conjugate in accordance withthe present invention. The polymer backbone can be linear, or may be inany of the above-described forms (e.g., branched, forked, and the like).Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), copolymers of ethylene glycol and propyleneglycol, poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(acrylic acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. In addition to PEG, poly(acrylic acid) is a preferredpolymer species because it has the known property of adherence tomucosa, which may offer advantages in binding of the conjugatedantagonist to membrane surface receptors.

B. Linkage Between Polymer Backbone and Opioid Antagonist

The linkage between the opioid antagonist and the polymer backbone(i.e., X in Formula I) results from the reaction of a reactivefunctional group of the polymer with a functional group on the opioidantagonist molecule. The specific linkage will depend on the structureof the functional groups utilized, and will typically be governed by thefunctional groups contained in the opioid antagonist molecule. Forexample, an amide linkage can be formed by reaction of a polymer havinga terminal carboxylic acid group, or an active ester thereof, with anopioid antagonist having an amine group. Alternatively, a sulfidelinkage can be formed by reaction of a polymer terminated with a thiolgroup with an opioid antagonist bearing a hydroxyl group. In anotherembodiment, an amine linkage is formed by reaction of anamino-terminated polymer with an opioid antagonist bearing a hydroxylgroup. The particular coupling chemistry employed will depend upon thestructure of the opioid antagonist, the potential presence of multiplefunctional groups within the opioid antagonist, the need forprotection/deprotection steps, chemical stability of the molecule, andthe like, and will be readily determined by one skilled in the art.Illustrative linking chemistry useful for preparing the polymerconjugates of the invention can be found, for example, in Wong, S. H.,(1991), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press,Boca Raton, Fla. and in Brinkley, M. (1992) “A Brief Survey of Methodsfor Preparing Protein Conjugates with Dyes, Haptens, and CrosslinkingReagent”s, in Bioconjug. Chem., 3, 2013.

The linkage is preferably hydrolytically stable to prevent release ofthe opioid antagonist after administration to the patient, therebyreducing the possibility of transport of the antagonist through theintestinal barrier and into the bloodstream. Once in the bloodstream,there is an increased possibility of the antagonist passing through theblood-brain barrier and negatively impacting the analgesic effect of theopioid, depending of course on the particulars of the particular opioidantagonist. Exemplary hydrolytically stable linkages include amide,amine, carbamate, ether, thioether, and urea.

The overall X linkage is intended to encompass any linkage between thepolymer and the opioid antagonist molecule having an overall length offrom 1 to about 20 atoms, preferably 1 to about 10 atoms.

In Formula Ia above, the X linkage is preferably a secondary amine oramide linkage. In one embodiment of Formula Ia, X has the formula—NH—(CHR₀)_(m)—O— or —NH—C(O)—(CHR₀)_(n)—O—, wherein m is 1-12,preferably 1-4 (i.e., 1, 2, 3, or 4) and each R₀ is independently H orC1-C6 alkyl (e.g., methyl or ethyl). In Formula Ib above, the X linkageis preferably a heteroatom, such as an ether or thioether linkage (i.e.,X═O or S).

C. Opioid Antagonists

As defined herein, an “opioid antagonist” is any molecule that blocksthe action of an opioid agonist at one or more opioid receptor types,including so-called “agonist-antagonist” molecules that act as anantagonist for one opioid receptor type and an agonist for anotherreceptor type (e.g., nalorphine or pentazocine). The opioid antagonistpreferably exhibits no agonist activity for any opioid receptor type andpreferably exhibits antagonist activity for μ-receptors. Many opioidantagonists are structurally similar to the closest agonist analogue,with the exception of a larger hydrocarbon group attached to the N₁₇position. For example, nalorphine is structurally identical to morphinewith the exception of replacement of the N₁₇ methyl group of morphinewith an allyl group. Suitable opioid antagonists include, but are notlimited to, buprenorphine, cyclazocine, cyclorphan, naloxone,N-methylnaloxone, naltrexone, N-methylnaltrexone, nalmephene,6-amino-6-desoxo-naloxone, levallorphan, nalbuphine, naltrendol,naltrindole, nalorphine, nor-binaltorphimine, oxilorphan, pentazocine,piperidine-N-alkylcarboxylate opioid antagonists such as those describedin U.S. Pat. Nos. 5,159,081; 5,250,542; 5,270,328; and 5,434,171 (all ofwhich are incorporated by reference herein), opioid antagonistpolypeptides (such as those described by R. J. Knapp, L. K. Vaughn, andH. I. Yamamura in “The Pharmacology of Opioid Peptides”, L. F. Tseng,Ed., p. 15, Harwood Academic Publishers, (1995)), and derivatives ormixtures thereof.

D. Method of Forming Polymer Conjugates of Opioid Antagonists

The polymer conjugate of the invention can be formed using knowntechniques for covalent attachment of an activated polymer, such as anactivated PEG, to a biologically active agent (See, for example,POLY(ETHYLENE GLYCOL) CHEMISTRY AND BIOLOGICAL APPLICATIONS, AmericanChemical Society, Washington, D.C. (1997)). The general method involvesselection of a reactive polymer bearing a functional group suitable forreaction with a functional group of the opioid antagonist molecule andreaction of the reactive polymer with the opioid antagonist in solutionto form a covalently-bound conjugate.

Selection of the functional group of the polymer will depend, in part,on the functional group on the opioid antagonist molecule. Thefunctional group of the polymer is preferably chosen to result information of a hydrolytically stable linkage between the opioidantagonist and the polymer. A polymer of the invention suitable forcoupling to an opioid antagonist molecule will typically have a terminalfunctional group such as the following: N-succinimidyl carbonate (seee.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmannet al. Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J.19:1177 (1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem.179:301 (1978)), succinimidyl propionate and succinimidyl butanoate(see, e.g., Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al., Makromol. Chem. 180:1381 (1979), succinimidyl ester(see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see,e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al.Eur. J. Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem,13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343(1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references are incorporated herein by reference.

In a particular embodiment exemplified in Examples 1-4, the ketone groupof naloxone or naltrexone is subjected to reductive amination to form anamino derivative of naloxone or naltrexone using methodology describedby Jiang, et al. (J. Med. Chem., 1977, 20:1100-1102). The aminoderivative is then reacted with (i) an aldehyde-terminated polymer inthe presence of a reducing agent to form a secondary amine linkage or(ii) an active ester-terminated polymer to form an amide linkage.

The polymer conjugate product may be purified and collected usingmethods known in the art for biologically active conjugates of thistype. Typically, the polymer conjugate is isolated by precipitationfollowed by filtration and drying.

E. Exemplary Conjugate Structures

More specific structural embodiments of the conjugates of the inventionwill now be described, all of which are intended to be encompassed bythe structure of Formula I above. The specific structures shown beloware presented as exemplary structures only, and are not intended tolimit the scope of the invention.

An embodiment of a linear polymer of the invention can be structurallyrepresented as shown below:

R—POLY-X-A_(o)  Formula V

wherein POLY is a water soluble and non-peptidic polymer backbone, R isa capping group or a functional group, and X and A_(o) are as definedabove. In a preferred embodiment, R is methoxy, POLY is poly(ethyleneglycol), X is a hydrolytically stable linkage such as amide, amine,carbamate, sulfide, ether, thioether, or urea, and A_(o) has thestructure shown in Formula Ia or Formula Ib above.

The R group can be a relatively inert capping group, such as alkoxy(e.g., methoxy or ethoxy), alkyl, benzyl, aryl, or aryloxy (e.g.,benzyloxy). Alternatively, the R group can be a functional group capableof readily reacting with a functional group on a biologically activemolecule such as another opioid antagonist. Exemplary functional groupsinclude hydroxyl, active ester (e.g. N-hydroxysuccinimidyl ester or1-benzotriazolyl ester), active carbonate (e.g. N-hydroxysuccinimidylcarbonate and 1-benzotriazolyl carbonate), acetal, aldehyde, aldehydehydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone,amine, hydrazide, thiol, carboxylic acid, isocyanate, isothiocyanate,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,epoxide, glyoxal, dione, mesylate, tosylate, or tresylate.

In a homobifunctional embodiment of Formula V, R has the structurewherein X and A_(o) are as defined above.

One example of a multi-arm embodiment of the polymer conjugate of theinvention has the structure:

wherein each POLY is a water soluble and non-peptidic polymer backbone,R′ is a central core molecule, y is from about 3 to about 100,preferably 3 to about 25, and X and A_(o) are as defined above. The coremoiety, R′, is a residue of a molecule selected from the groupconsisting of polyols, polyamines, and molecules having a combination ofalcohol and amine groups. Specific examples of central core moleculesinclude glycerol, glycerol oligomers, pentaerythritol, sorbitol, andlysine.

The central core molecule is preferably a residue of a polyol having atleast three hydroxyl groups available for polymer attachment. A “polyol”is a molecule comprising a plurality of available hydroxyl groups.Depending on the desired number of polymer aims, the polyol willtypically comprise 3 to about 25 hydroxyl groups. The polyol may includeother protected or unprotected functional groups as well withoutdeparting from the invention. Although the spacing between hydroxylgroups will vary from polyol to polyol, there are typically 1 to about20 atoms, such as carbon atoms, between each hydroxyl group, preferably1 to about 5. Preferred polyols include glycerol, reducing sugars suchas sorbitol, pentaerythritol, and glycerol oligomers, such ashexaglycerol. A 21-arm polymer can be synthesized usinghydroxypropyl-β-cyclodextrin, which has 21 available hydroxyl groups.The particular polyol chosen will depend on the desired number ofhydroxyl groups needed for attachment to the polymer arms. In thisparticular embodiment, as the number of polymer arms in the conjugate isincreased, the molecular weight or number of monomer subunits of each ofthe polymer arms will preferably decrease in order to keep within thepreferred molecular weight ranges for a conjugate in accordance with theinvention.

III. PHARMACEUTICAL COMPOSITIONS INCLUDING A POLYMER CONJUGATE OF THEINVENTION

The invention provides pharmaceutical formulations or compositions, bothfor veterinary and for human medical use, which comprise one or morepolymer conjugates of the invention or a pharmaceutically acceptablesalt thereof, with one or more pharmaceutically acceptable carriers, andoptionally any other therapeutic ingredients, stabilizers, or the like.The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof. The compositions of the inventionmay also include polymeric excipients/additives or carriers, e.g.,polyvinylpyrrolidones, derivatized celluloses such ashydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin),polyethylene glycols, and pectin. The compositions may further includediluents, buffers, binders, disintegrants, thickeners, lubricants,preservatives (including antioxidants), flavoring agents, taste-maskingagents, inorganic salts (e.g., sodium chloride), antimicrobial agents(e.g., benzalkonium chloride), sweeteners, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”, andpluronics such as F68 and F88, available from BASF), sorbitan esters,lipids (e.g., phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines, fatty acids and fattyesters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA,zinc and other such suitable cations). Other pharmaceutical excipientsand/or additives suitable for use in the compositions according to theinvention are listed in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andin “Handbook of Pharmaceutical Excipients”, Third Ed., Ed. A.H. Kibbe,Pharmaceutical Press, 2000.

The conjugates of the invention may be formulated in compositionsincluding those suitable for oral, rectal, topical, nasal, ophthalmic,or parenteral (including intraperitoneal, intravenous, subcutaneous, orintramuscular injection) administration. The compositions mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing the active agent or compound (i.e., the polymerconjugate) into association with a carrier that constitutes one or moreaccessory ingredients. In general, the compositions are prepared bybringing the active compound into association with a liquid carrier toform a solution or a suspension, or alternatively, bring the activecompound into association with formulation components suitable forforming a solid, optionally a particulate product, and then, ifwarranted, shaping the product into a desired delivery form. Solidformulations of the invention, when particulate, will typically compriseparticles with sizes ranging from about 1 nanometer to about 500microns. In general, for solid formulations intended for intravenousadministration, particles will typically range from about 1 nm to about10 microns in diameter.

The amount of polymer conjugate in the formulation will vary dependingupon the specific opioid antagonist employed, its activity in conjugatedform, the molecular weight of the conjugate, and other factors such asdosage form, target patient population, and other considerations, andwill generally be readily determined by one skilled in the art. Theamount of conjugate in the formulation will be that amount necessary todeliver a therapeutically effective amount of opioid antagonist to apatient in need thereof to achieve at least one of the therapeuticeffects associated with the opioid antagonist, e.g., relief of one ormore side effects of opioid use, such as nausea, constipation orpruritus. In practice, this will vary widely depending upon theparticular conjugate, its activity, the severity of the condition to betreated, the patient population, the stability of the formulation, andthe like. Compositions will generally contain anywhere from about 1% byweight to about 99% by weight conjugate, typically from about 2% toabout 95% by weight conjugate, and more typically from about 5% to 85%by weight conjugate, and will also depend upon the relative amounts ofexcipients/additives contained in the composition. More specifically,the composition will typically contain at least about one of thefollowing percentages of conjugate: 2%, 5%, 10%, 20%, 30%, 40%, 50%,60%, or more by weight.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,lozenges, and the like, each containing a predetermined amount of theactive agent as a powder or granules; or a suspension in an aqueousliquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, adraught, and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which is optionally mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent or dispersing agent. Molded tablets comprised with a suitablecarrier may be made by molding in a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredient(s). Such accessory ingredients mayinclude flavorings, suitable preservatives, an agent to retardcrystallization of the sugar, and an agent to increase the solubility ofany other ingredient, such as polyhydric alcohol, for example, glycerolor sorbitol.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the conjugate, which can beformulated to be isotonic with the blood of the recipient.

Nasal spray formulations comprise purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, or hydrogenated fats orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the active compound dissolved or suspendedin one or more media such as mineral oil, petroleum, polyhydroxyalcohols or other bases used for topical formulations. The addition ofother accessory ingredients as noted above may be desirable.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired polymer conjugate or a saltthereof. The desired formulation may be placed in a small chamber andnebulized. Nebulization may be accomplished by compressed air or byultrasonic energy to form a plurality of liquid droplets or solidparticles comprising the conjugates or salts thereof.

IV. METHOD OF USING THE POLYMER CONJUGATES

The polymer conjugates of the invention can be used to treat anycondition responsive to opioid antagonists in any animal, particularlyin mammals, including humans. A preferred condition for treatment is anyside effect associated with opioid analgesic use, such as nausea,constipation, or pruritus. Alternatively, the polymer conjugate of theinvention can be used prophylactically to prevent the side effects ofopioid use. The method of treatment comprises administering to themammal a therapeutically effective amount of a composition orformulation containing a polymer conjugate of an opioid antagonist asdescribed above. The therapeutically effective dosage amount of anyspecific conjugate will vary somewhat from conjugate to conjugate,patient to patient, and will depend upon factors such as the conditionof the patient, the activity of the particular opioid antagonistemployed, and the route of delivery. As would be understood, if apolymer conjugate of an opioid antagonist has reduced antagonistactivity as compared to the unconjugated parent molecule, higher dosescan be used to offset the reduced activity. As a general proposition, adosage from about 0.5 to about 100 mg/kg body weight, preferably fromabout 1.0 to about 20 mg/kg, will have therapeutic efficacy. Whenadministered conjointly with other pharmaceutically active agents, evenless of the polymer conjugate may be therapeutically effective.

The polymer conjugate may be administered once or several times a day.The duration of the treatment may be once per day for a period of fromtwo to three weeks and may continue for a period of months or evenyears. The daily dose can be administered either by a single dose in theform of an individual dosage unit or several smaller dosage units or bymultiple administration of subdivided dosages at certain intervals.

The polymer conjugate can be administered conjointly with the opioidagonist, meaning the polymer conjugate and the opioid agonist areadministered at the same time or the opioid agonist and the polymerconjugate are both administered within a short time interval in anyorder. Preferably, the opioid agonist and the polymer conjugate areadministered at the same time or within about an hour apart, morepreferably within about 30 minutes apart, even more preferably withinabout 15 minutes apart (in any order). As would be understood in theart, if the opioid agonist and the polymer conjugate are administeredconjointly, the two therapeutic agents can be administered in the sameformulation (i.e., in the same dosage unit).

Oral delivery is the preferred route of administration for both theopioid agonist and the polymer conjugate of the invention. However, boththerapeutic agents could be delivered using other routes and differentroutes of administration could be used for each therapeutic agent. Forexample, the opioid agonist could be delivered intravenously and thepolymer conjugate of the invention could be delivered orally.

As used herein, an “opioid agonist” is any natural or synthetic alkaloidof opium that activates one or more opioid receptor types, includingpartial agonists (i.e., compounds exhibiting activity against less thanall opioid receptor types) and agonist-antagonists (i.e., compoundsexhibiting agonist activity at one receptor type and antagonist activityat another receptor type). The opioid agonist can be a natural alkaloidsuch as a penanthrene (e.g., morphine) or benzylisoquinoline (e.g.,papaverine), a semi-synthetic derivative (e.g., hydromorphone), or anyof various classes of synthetic derivatives (e.g., phenylpiperidines,benzmorphans, priopionanilides, and morphinans). Exemplary opioidagonists include alfentanil, bremazocine, buprenorphine, butorphanol,codeine, cyclazocine, dezocine, diacetylmorphine (i.e., heroin),dihydrocodeine, fentanyl, hydrocodone, hydromorphone, levorphanol,meperidine (pethidine), methadone, morphine, nalbuphine, noscapine,oxycodone, oxymorphone, papaverine, pentazocine, pethidine, phenazocine,propiram, propoxyphene, sufentanil, thebaine and tramadol. Preferably,the opioid agonist is selected from the group consisting of morphine,codeine, oxycodone, hydrocodone, dihydrocodeine, propoxyphene, fentanyl,and tramadol.

V. EXAMPLES

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention. For example, althoughmPEG is used in the examples to illustrate the invention, other forms ofPEG and similar polymers that are useful in the practice of theinvention are encompassed by the invention as discussed above.

All PEG reagents referred to in the appended examples are available fromShearwater Corporation of Huntsville, Ala. All ¹H NMR data was generatedby a 3 or 400 MHz NMR spectrometer manufactured by Bruker.

Examples 1-4 illustrate methods of forming polymer conjugates using mPEGas the polymer backbone and 6-amino-6-desoxo-naloxone as the opioidantagonist. Examples 1 and 3 illustrate the formation of ahydrolytically stable secondary amine linkage between the polymerbackbone and the opioid antagonist. Examples 2 and 4 illustrate theformation of a hydrolytically stable amide linkage between the polymerbackbone and the opioid antagonist. Note that 6-amino-6-desoxo-naloxoneexists as a mixture of two epimers, alpha and beta. Both epimers arebelieved to be active and, thus, a mixture of the two epimers can beused. However, although not exemplified below, the two epimers can beseparated using separation methods known in the art.

Example 1 Preparation of 6-mPEG(550 Da)-NH-6-desoxo-naloxone (mixture of6-epimers) A. Synthesis of 6-amino-6-desoxo-naloxone (mixture of 6-aminoepimers)

Naloxone was subjected to reductive amination by methods similar tothose of Jiang, et al. (J. Med. Chem., 20: 1100-1102, 1977). To amixture of naloxone (7.4 g) and ammonium acetate (15.4 g) dissolved inmethanol (50 ml) under nitrogen was added a methanolic solution(40 nil)of NaCNBH₃ (1.4 g). The resulting solution was adjusted to pH 7.0 withconcentrated HCl, stirred for 20 hours, and acidified to pH 1 withaddition of concentrated HCl. After removal of the solvent anddissolution of the residue in water, the aqueous solution was extractedwith chloroform to remove the water-insoluble material and was thenadjusted to pH 9.0 with Na₂CO₃. The mixture was saturated with NaCl andextracted with CHCl₃. The CHCl₃ phase was dried with Na₂SO₄ andevaporated to dryness. The oily residue was dissolved in 60 ml ofmethanol, acidified to pH 1.0 with concentrated HCl and allowed to standovernight overnight at 4° C. The solvent was evaporated to dryness andthe residue dried under vacuum.

Yield: 7.13 g. ¹H nmr (DMSO-d₆): δ 6.56 ppm and δ 6.52 ppm (1H each, twodoublets, aromatic H), δ 5.83 ppm (1H multi. olefinic H), δ 5.18 ppm (2Hmulti. olefinic H), S 5.01 ppm (1H singlet,), δ 4.76 ppm (1H singlet).

B. Preparation of 6-mPEG(550 Da)-NH-6-desoxo-naloxone (mixture of6-epimers)

To a mixture of mPEG-550-aldehyde (Shearwater Corporation, M.W. 550 Da2.0 g, 3.3 mmol) and 6-NH₂-naloxone 2HCl (1.6 g, 4.0 mmol) (from Step A)dissolved in deionized water (25 ml) under argon was added an aqueoussolution (20 ml) of NaCNBH₃ (0.15 g, 2.4 mmol). The resulting solutionwas stirred at room temperature under argon overnight (18 h). Thesolution was then diluted with DI water (350 ml), acidified withconcentrated HCl to pH 1 and washed with CHCl₃ (3×150 ml) to removeunbound PEG. To the aqueous phase was added Na₂HPO₄ (6.0 g, 42 mmol,˜100 mM), the pH adjusted to 6.0 with NaOH and the resulting solutionextracted with CHCl₃ (3×200 ml). The CHCl₃ extracts were combined,washed with a pH 6.5 phosphate buffer (100 mM, 3×200 ml), dried overanhydrous Na₂SO₄, evaporated under vacuum and dried in vacuo for 2 days.The pure conjugate was obtained as a light yellow liquid (1.3 g, 1.4mmol, 42% yield).

¹H NMR (CDCl₃): δ 6.51-6.74 (2H, multiplet, aromatic protons ofnaloxone); 5.72-5.85 (1H, multiplet, olefinic proton of naloxone); 5.16(2H, triplet, olefinic protons of naloxone); 4.76 and 4.46 (1H, twodoublet, C₅ proton of α and β naloxone); 3.64 (˜57H, multiplet, PEG);3.38 (3H, singlet, methoxy protons of PEG); 1.34-3.12 (20H, multiplet,protons of naloxone and PEG) ppm.

Example 2 Preparation of 6-mPEG (550 Da)-CONH-6-desoxo-naloxone (mixtureof 6-epimers)

mPEG(550) N-succinimidyl propionate (Shearwater Corporation, 4.0 g, 5.5mmol) and 6-amino-6-desoxo-naloxone (2.0 g, 6.1 mmol) (from Step A ofExample 1) were dissolved in CHCl₃ (50 ml) under argon. The solution wasstirred at room temperature under argon overnight (20 h). CHCl₃ (250 ml)was added and the solution was extracted with pH 1HCl solution (3×200ml). To the combined aqueous extracts was and extracted with CHCl₃(3×200 ml). The CHCl₃ extracts were combined, washed with a pH 5.5phosphate buffer solution (50 mM, 3×200 ml), dried over anhydrous Na₂SO₄and filtered. All solvents were removed with a rotary evaporator and theresulting product was dried in vacuo for 2 days to give purem-PEG-550-CONH-Naloxone conjugate as a colorless liquid (3.5 g, 3.7mmol, 66% yield).

¹H NMR (CDCl₃): δ 7.12 and 6.88 (1H, two doublet, NHCO of α and βconjugates); 6.50-6.71 (2H, multiplet, aromatic protons of naloxone);5.72-5.87 (1H, multiplet, olefinic proton of naloxone); 5.17 (2H,triplet, olefinic protons of naloxone); 4.58 and 4.40 (1H, two doublet,C₅ proton of α and β naloxone); 3.64 (−54H, multiplet, PEG); 3.38 (3H,singlet, methoxy protons of PEG); 0.83-3.13 (14H, multiplet, protons ofnaloxone) ppm.

Example 3 Synthesis of mPEG (2000 Da)-6-desoxo-naloxone

To a mixture of 6-amino-6-desoxonaloxone.2HCl (0.6 g) (from Step A ofExample 1) and mPEG (2000 Da)-propionaldehyde (6.0 g) dissolved in 0.1 Mphosphate buffer pH 6.5 was added phosphate buffer solution (pH 6.5, 5ml) of NaCNBH₃. The resulting solution was stirred at room temperatureunder argon overnight. The reaction mixture was diluted to 500 ml,saturated with NaCl and extracted with dichloromethane. The extracteddichloromethane was dried with Na₂SO₄, evaporated and precipitated withethyl ether. The product was dried under vacuum overnight. Yield: 5.63 gGPC: ˜25% of conjugates.

The mixture product was purified through cation exchange chromatographyusing Poros 50 HS resin (100 ml). The mixture was dissolved in 200 ml ofdeionized water and was loaded on cation exchange column (3.5×28 cm).After column was washed with 500 ml of deionized water, 1N NaCl solution(500 ml) was used to elute the column. The desired conjugate wasobtained after extraction with DCM, evaporation and precipitation withEt₂O. Yield: ˜1.38 g.

The conjugate was further purified by reverse phase HPLC chromatography(Betasil C18 column, Keystone Scientific).

Example 4 Synthesis of mPEG (2000 Da)-6-desoxo-naloxone

mPEG(2000 Da)-N-succinimidyl propionate (5.0 g) was dissolved in 50 mlof dichloromethane. 1.88 g of 6-amino-naloxone 2HCl (from Step A ofExample 1) and 1.4 ml of triethylamine were added to the solution. Theresulting solution was stirred at room temperature under argonovernight. The reaction mixture was filtered and the filtrate evaporatedand precipitated with isopropanol/diethyl ether. The product was driedunder vacuum overnight. It was then redissolved in 500 ml of deionizedwater, adjusted to pH to 9.0 with 1 N NaOH, saturated with NaCl, washedwith diethyl ether, and finally extracted with dichloromethane. Theextracted dichloromethane was dried with Na₂SO₄, the solvent removedunder vacuum and the product precipitated from Et₂O. The product wasdried under vacuum overnight.

Yield: 3.6 g. ¹H nmr (DMSO-d₆): δ 8.08 ppm and 7.53 ppm (1H, twodoublets, amide H), δ 6.60-5.45 ppm (2H multi., aromatic H), δ 5.83 ppm(1H multi. olefinic H), δ 5.25-5.12 ppm (2H multi. olefinic H). δ 4.76ppm (1H singlet). GPC: 97% conjugation. HPLC: showed no freeamino-naloxone.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

1.-26. (canceled)
 27. A method of treating constipation resulting fromthe administration of an opioid agonist to a patient in need thereof,said method comprising administering to the patient in need thereof atherapeutically effective amount of a polymer conjugate of the formula:

or pharmaceutically acceptable salt thereof, wherein: Y is allyl; Z isOH; X is —NHC(O)—, —NH—, —OC(O)NH —, —O—, —S— or —NHC(O)NH—; and POLY ismethoxy poly(ethylene glycol), wherein POLY has a molecular weight offrom about 100 Da to about 2,000 Da.
 28. A method of treating nausearesulting from the administration of an opioid agonist to a patient inneed thereof, said method comprising administering to the patient inneed thereof a therapeutically effective amount of a polymer conjugateof the formula:

or pharmaceutically acceptable salt thereof, wherein: Y is allyl; Z isOH; X is —NHC(O) —, —NH—, —OC(O)NH —, —O—, —S— or —NHC(O)NH—; and POLYis methoxy poly(ethylene glycol), wherein POLY has a molecular weight offrom about 100 Da to about 2,000 Da.
 29. A method of treating pruritusresulting from the administration of an opioid agonist to a patient inneed thereof, said method comprising administering to the patient inneed thereof a therapeutically effective amount of a polymer conjugateof the formula:

or pharmaceutically acceptable salt thereof, wherein: Y is allyl; Z isOH; X is —NHC(O) —, —NH—, —OC(O)NH—, —O—, —S— or —NHC(O)NH—; and POLY ismethoxy poly(ethylene glycol), wherein POLY has a molecular weight offrom about 100 Da to about 2,000 Da.
 30. The method of claim 27, whereinPOLY has the formulaCH₃O—(CH₂CH₂O)_(n)—CH₂CH₂— wherein n is from 2 to
 45. 31. The method ofclaim 30, wherein n is from 2 to
 20. 32. The method of claim 27, whereinPOLY has a molecular weight of from about 100 Da to about 500 Da. 33.The method of claim 27, wherein X is —NHC(O)
 34. The method of claim 27,wherein X is —NH—.
 35. The method of claim 27, wherein X is —OC(O)NH—.36. The method of claim 27, wherein X is —O—.
 37. The method of claim27, wherein X is —S—.
 38. The method of claim 27, wherein X is—NHC(O)NH—.